MEMBRANE POTENTIALS AND pH GRADIENTS IN MICROSCOPIC SYSTEMS: THE CHEMIOSMOTIC PARADIGM H'R' Kaback

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MEMBRANE POTENTIALS AND pH
GRADIENTS
IN MICROSCOPIC SYSTEMS THE CHEMIOSMOTIC
PARADIGMH.R. Kaback
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A Little History In 1960-61, the British
scientist Peter Mitchell put forward the
heretical postulate that a pH gradient (the
so-called Proton-Motive Force) across the
mitochondrial or bacterial membrane is the
immediate driving force for oxidative
phosphorylation, as well as the accumulation of
metabolites against a concentration gradient (The
Chemiosmotic Hypothesis). At
about the same time, Robert Crane of Rutgers
University suggested that sodium gradients are
responsible for driving glucose accumulation
across the intestinal epithelium.
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In the late 60s, Andre Jagendorf of Cornell
University carried out the first experiments that
made scientists begin to take Mitchells
hypothesis seriously. By using thylakoids (the
intracellular organelles responsible for
photophosphorylation in plants), Jagendorf
demonstrated that sudden acidification of the
external medium leads to synthesis of ATP. Over
the ensuing 10 years, work in many laboratories
using a variety of techniques with different
experimental systems showed virtually
unequivocally that Mitchells Chemiosmotic
Hypothesis is the paradigm for bioenergetics in
energy transducing membranes, and in 1977,
Mitchell was awarded the Nobel Prize in
Chemistry.
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Clearly at this level, the major questions are
two-fold a. Are membrane potentials and pH
gradients present in microscopic systems (too
small to be impaled by microelectrodes)? b. If
there are membrane potentials and pH gradients in
these systems, are they of sufficient magnitude
to drive the process in question? In this
lecture, the focus is on right-side-out (RSO)
membrane vesicles from E. coli and
respiration-driven active transport in this
well-defined system with the goal of providing a
basic intuitive understanding of the Chemiosmotic
Paradigm with respect to active transport. We
will then see how the principles apply to more
complex eucaryotic systems.
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Although there are instances in which loops
probably occur in respiratory chains, things are
generally more complicated. For example, certain
terminal oxidases in purified form pump protons
by themselves when reconstituted into artificial
membranes. There are also respiratory chains in
some bacteria that pump sodium. The point is
that loops in the respiratory chain provide a
simple conceptual possibility for how electron
transfer might be coupled to proton pumping.
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Figure 3
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Figure 4
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From the decrease in the dialyzable 14Cacetate
concentration upon addition of ASC-PMS, the
quantity of acetate accumulated by the vesicles
can be readily calculated per mg of vesicle
protein, and knowing the intravesicular volume
per mg of vesicle protein, this value can be
transformed into the internal concentration of
14Cacetate (in molarity). Since the external
concentration of the weak acid in the medium
surrounding the vesicles is given directly from
the flow dialysis profile, the concentration
gradient of 14Cacetate in the presence of
ASC-PMS is determined from acetatein/acetateou
t and converted in mV by using the constants
shown in Figure 1 (2.3RT/F?60 at room
temperature).
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